Microbial infections represent a major cause of morbidity and mortality worldwide, and the spectrum of microorganisms causing disease continues to increase. Microorganisms (bacteria, fungi and yeast) responsible for causing infectious diseases are traditionally detected in hospital laboratories with the aid of microbiological culture methods with poor sensitivity (25-82%), which are very time-consuming, generally taking from two to five days to complete, and up to eight days for the diagnosis of fungal infections. Definitive diagnosis is usually based on either, the recovery and identification of a specific microorganism from clinical specimens or microscopic demonstration of fungi with distinct morphological features. However, there are numerous cases where these methods fail to provide conclusive proof as to the infecting agent or microrganism. In these instances, the detection of specific host antibody responses can be used, although again this can be affected by the immune status of the patient.
Time is critical in the detection and identification of infectious microorganisms. Effective treatment depends on finding the source of infection and making appropriate decisions about antibiotics quickly and efficiently. Only after pathogens are correctly identified, can targeted therapy using a specific antibiotic begin. Many physicians would like to see the development of better in vitro amplification and direct detection diagnostic techniques for the early diagnosis of microbial infection. Recently, Roche™ launched a real time PCR based assay (Septifast™), for the detection of microbial DNA in clinical samples. Therefore, there is a clear need for the development of novel rapid diagnostic tests for clinically significant bacterial and fungal pathogens for bioanalysis applications in the clinical sector. This has led the current inventors to identify novel nucleic acid targets for application in Nucleic Acid Diagnostic (NAD) tests.
It is clear though, that development of faster, more accurate diagnostic methods are required, particularly in light of the selection pressure caused by modern anti-microbial treatments which give rise to increased populations of resistant virulent strains with mutated genome sequences. Methods that enable early diagnosis of microbial causes of infection enable the selection of a specific narrow spectrum antibiotic or antifungal to treat the infection (Datamonitor report: Stakeholder opinion—Invasive fungal infections, options outweigh replacements 2004; Datamonitor report: Stakeholder Opinion-Sepsis, under reaction to an overreaction, 2006).
LepA (leader peptidase A) has recently been assigned the function of ribosomal elongation factor (Qin et al., 2006, Cell). LepA is highly conserved and is present in all bacteria and mitochondria. There are 2444 LepA gene sequences (˜1.8 kb in length) available in GenBank including 2229 bacterial sequences. Using Clustal W sequence alignments, the LepA gene of Bacillus, Listeria, Enterobacteriaceae, Mycobacteria, Staphylococci and Streptococci were compared in silico to other molecular targets including tufA and the ssrA genes. In general, LepA seemed to have sufficient sequence heterogeneity to enable its application for microorganism species identification in nucleic acid based tests (Table 1).
TABLE 1Percentage range of homology between Bacillusspecies, Listeria species, Enterobacteriaceae, Mycobacteriumspecies, Streptococcus species and Staphylococcus speciesin the LepA gene compared to the tufA (equivalent commercialisedmRNA) and ssrA genes (RiboSEQ technology).LepA (rangetufA (rangessrA (rangeof % homol-of % homol-of % homol-ogy betweenogy betweenogy betweenspecies)species)species)Bacillus species72-9781-9962-100Listeria species89-909997-99 Enterobacteriaceae59-9983-9992-99 (including E. coli)Mycobacterium species78-99 87-10084-100Streptococcus species70-9176-9762-100Staphylococcus species80-8391-9581-99 
GUF1, which is similar to the E. coli elongation factor-type GTP-binding protein LepA, is a gene encoding a novel evolutionarily conserved GTPase coding protein (GTPase of Unknown Function 1, Kiser G L and Weinert T A (1995) GUF1, a gene encoding a novel evolutionarily conserved GTPase in budding yeast. Yeast 11(13): 1311-6), which, was predicted to be the GTPase of the elongation factor-type class. There are 94 Guf1 sequences available in NCBI GeneBank including 3 Candida and 6 Aspergillus. 